Research in my laboratory focuses on the understanding of the mechanisms of bacterial pathogenesis, antibiotic resistance of gram-negative bacteria, and the development of novel antimicrobial agents and vaccines, specifically as they relate to ulcer disease and other bacterial infections. Gram-negative bacteria (pathogens and nonpathogens) have a unique structure called the outer membrane that makes these bacteria more refractory to antibiotic therapy than their gram-positive counterparts (save mycobacteria). Research findings in my laboratory provided the genetic, biochemical, and structural evidence for the role of ADP-L-glycero-D-mannoheptose 6-epimerase (epimerase) in the synthesis of the lipopolysaccharide precursor L-glycero-D-mannoheptose (heptose) in several genera of gram-negative bacteria. These findings are relevant to the management of infection, taking advantage of the observations that gram-negative bacteria with defective heptose biosynthesis have altered growth rate, virulence, increased susceptibility to antibiotics and that heptose is not found in mammalian cells. Therefore, the heptose biosynthetic enzymic steps in bacteria are unique targets for the design of novel antimicrobial agents. Our structural studies of epimerase (done in collaboration with Drs. Steve E. Ealick and Ashley M. Deacon) indicated a high structural similarity to the short-chain dehydrogenases/reductases superfamily and the presence of the conserved catalytic motif TyrXXXLys. We have completed mutagenic and kinetic studies to confirm a role for Ser116, Tyr140 and Lys144 residues in ADP-L-glycero-D-mannoheptose 6-epimerase catalysis. In collaboration, with Dr. Martin Tanner?s group at the University of British Columbia, we further expanded research in the catalytic mechanism of ADP-L-glycero-D-mannoheptose 6-epimerase catalyzed reactions. Epimerase is known to require a tightly bound NADP+ cofactor for activity and presumably employs a mechanism involving transient oxidation of the substrate. Four mechanistic possibilities are considered that involve transient oxidation at either C-7", C-6" or C-4" of the heptose nucleotide. In our recent paper, we provided strong evidence for a mechanism involving non-stereospecific oxidation/reduction directly at C-6" based on experiments with solvent isotope incorporation and alternate substrates. The results showed that epimerization proceeds without any detectable incorporation of solvent-derived deuterium or 18O-isotope into the product. This argues against mechanisms involving either proton transfers at carbon or dehydration/rehydration events. In addition, the deoxygenated analogs, 7"-deoxy-ADP-L, D-Hep and 4"-deoxy-ADP-L, D-Hep, were both found to serve as substrates for the enzyme, indicating that oxidation at either C-7" or C-4" is not required for catalysis. In another collaborative study with Dr. Martin Tanner?s group, we provided the first positive evidence for the utilization of a direct C-6' ' oxidation/reduction mechanism by ADP-L-glycero-D-manno-heptose 6-epimerase (epimerase). The stereochemical inversion catalyzed by the epimerase is interesting as it occurs at an "unactivated" stereocenter that lacks an acidic C-H bond, and therefore, a direct deprotonation/reprotonation mechanism cannot be employed. Instead, the epimerase employs a transient oxidation strategy involving the tightly bound NADP(+) cofactor. In this study, a reaction intermediate analogue containing an aldehyde functionality at C-6' ', ADP-beta-d-manno-hexodialdose, was prepared and used to probe the ability of the enzyme to catalyze redox chemistry at this position. It was found that incubation of the aldehyde with a catalytic amount of the epimerase leads to a dismutation process in which one-half of the material is oxidized to ADP-beta-d-mannuronic acid and the other half is reduced to ADP-beta-d-mannose. Transient reduction of the enzyme-bound NADP(+) was monitored by UV spectroscopy and implicates the cofactor's involvement during catalysis. We have extended our investigation of the outer membrane to the gram-negative strain, Helicobacter pylori. H. pylori is the causative agent for gastritis, ulcer disease and some gastric cancers. The mechanism of H. pylori pathogenesis is not known at this time. We are using a mouse model for the study of H. pylori infection, detection methods, pathogenic mechanism(s) and, an in vivo expression technology system to identify a subset of genes induced during infection. We have developed a non-invasive, sensitive and species-specific method to detect H. pylori infection of mice by PCR analysis of fecal pellets extracts. The extension of these findings to human subjects application is the subject of a pending patent. Another ongoing project is designed to identify and characterize novel targets for the development of antibiotics and protective vaccines directed against H. pylori. Two LPS core biosynthetic genes (rfaD and rfaE) from H. pylori have been cloned and the gene products purified. Studies to characterize the rfaE gene product, ADP-D-glycero-D-mannoheptose synthetase (the native enzyme has a molecular mass of 312 kDa and a subunit molecular weight of 52,000), from Helicobacter pylori are ongoing. To study host requirements for establishing successful infection and subsequent pathogenesis, we infected mutant C57BL/6 mice with genetic alterations that may influence the virulence of H. pylori. For this study two C57BL/6 mutant mouse strains were selected: an interleukin 10-deficient (IL-10) knockout and p47phox knockout strain. Using these knockout mice, we are evaluating the role of endogenous IL-10 on the regulation of the immune response to H. pylori infection. The p47phox knockout mice allow us to examine the in vivo role of NADP oxidase mediated inflammatory responses to Helicobacter pylori infection and pathology. We are initiating a study to compare immune responses to Helicobacter pylori infection involving a common adaptor molecule, myeloid differentiation protein MyD88 and selected Toll-like receptors in MyD88-/-, TLR2-/-, and TLR4-/- knockout animals. Our goal is to assess the relative contribution of MyD88 and Toll-like receptors TLR2 and TLR4 in the host?s response to H. pylori infection. We are also studying pathogenic mechanism(s) of H. pylori using mouse macrophage cell lines RAW 264.7 and J774.1A as well as primary dendritic cells from several knockout mice. Our preliminary fluorescence and confocal microscopic studies demonstrate that H. pylori can infect macrophage cells and can survive intercellularly for 24 hours. In summary, both the E. coli and H. pylori studies should advance our understanding of LPS core synthesis, gram-negative bacterial infection, and pathogenesis and these serve as basis for infection control and management protocols.